Pro-inflammatory role of megakaryocytes in arthritis Project Summary In inflammatory arthritis, the joints become the focus of intense immunological attack, frequently leading to irreversible injury and long-term disability. Multiple cell types hav been shown to participate in this process, including neutrophils, macrophages, synovial fibroblasts, mast cells, and most recently platelets. Here we present evidence implicating another lineage, the megakaryocyte. Megakaryocytes are hematopoietic cells found principally in the bone marrow, where they release large membrane-encapsulated cytoplasmic fragments that mature into normal platelets. Recently, megakaryocytes have been shown to release an abundance of much smaller (<1mm diameter) cytoplasmic fragments, termed megakaryocyte microparticles. These microparticles circulate in normal blood, suggesting the possibility that megakaryocytes may exert a physiological reach to distant tissues by mechanisms beyond release of platelets. However, as yet no such activity has been demonstrated in any system. The present submission provides evidence linking megakaryocytes directly to murine experimental arthritis. Among mature hematopoietic lineages, the stem cell factor/KitL receptor Kit is expressed most prominently by mast cells and megakaryocytes. Correspondingly, W/Wv mice bearing defective Kit display a lack of mast cells and a paucity of megakaryocytes. These animals are resistant to K/BxN serum transfer arthritis, a defect that can be corrected by mast cell engraftment. However, we find that arthritis resistance can also be repaired by a marrow lineage that expresses Kit, IL-1, and the platelet/megakaryocyte-specific collagen receptor glycoprotein VI (GPVI). Since W/Wv platelets are normal in number and function, and since adoptive transfer of platelets fails to repair arthritis susceptibility, we propose the novel and important hypothesis that megakaryocytes participate in the genesis of inflammatory arthritis above and beyond elaboration of platelets, whether via microparticles, cytokines, or other mechanism to be determined. To address this hypothesis, we outline two independent but complementary Aims. First, we will employ cultured murine megakaryocytes to assess phenotypic changes, microparticle production, and cytokine release in response to stimulation via Kit, GPVI and FcgRIII receptors. Further, we will characterize the IL-1 content of megakaryocyte microparticles as well as determine their pro-inflammatory potency in a fibroblast co-culture system. In a second Aim, we will employ established and novel approaches to confirm the in vivo relevance of MK to K/BxN serum transfer arthritis. Together, these studies have the potential to identify a previously unrecognized cellular contributor to synovitis and to begin to unravel the mechanisms underlying the ability of megakaryocytes to participate in a systemic inflammatory disease.